Viruses replicate and spread through infection of a host organism. For disease-causing viruses, controlling the spread of a viral infection is cause for global concern. Early detection of a viral infection is key to effectively treating infected patients and preventing epidemic spread.
The incubation period for many viruses is on the order of days to weeks and the infection period often begins before symptoms are present. It is therefore often challenging to prevent patient-to-patient infection without sensitive methods to detect viral infection before symptoms appear. Additionally, the effectiveness of many of the available antiviral medications rely on early detection and treatment of disease.
Although technology exists for detection of viral infection, the sensitivity is limited and most rely on sophisticated laboratory equipment and trained technicians. Thus in order to reduce the number of viral infections and improve early treatment, technology is needed for rapid point-of-care viral infection detection by individuals with little medical training.
Current methods for the detection of viral infections in patients rely primarily on “wet” lab techniques, which require an appropriately equipped laboratory and trained staff. Turnaround time for even the most rapid tests is often hours or days, limiting their use as point-of-care viral detection methods. Two commonly used techniques are enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).
ELISA tests detect virus in biological samples through the use of antibodies that bind viral antigens. Viral particles are adhered to a solid surface such as a plastic 96-well plate and are subsequently bound by antibodies that recognize a viral antigen. These primary antibodies or secondary antibodies are covalently linked to a fluorescent molecule or enzyme that produces colorimetric or luminescent signal upon exposure to a ligand. This signal is measured by a fluorimeter or spectrophotometer. ELISA can also be adapted to detect antibodies developed against virus in patient samples, however antibodies against virus are produced later in the infection process.
Although some ELISA methods are considered highly sensitive and can be quantitative, they require expensive laboratory equipment and their reproducibility is dependent on a well-trained technician. They are not suitable for rapid point-of-care detection and often require several hundred microliters of sample. Additionally, there is often disagreement over the numerical cutoff value of the quantified signal leading to potential false positives and false negatives.
PCR and reverse transcription PCR (RT-PCR) detect the nucleic acid component, i.e., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), of the virus and are generally considered to be more sensitive than ELISA with fewer false positives. PCR uses RNA primers designed to match known sequences in the viral RNA or DNA to amplify fragments which are used as a readout for presence of the virus. PCR and RT-PCR can be quantitative, allowing for accurate calculation of viral load. However, both PCR-based methods require sophisticated and expensive laboratory equipment and trained technicians, eliminating their use as rapid point-of-care tests.
A handful of rapid point-of-care diagnostic tests exist, with the two most widely used being for the detection of human immunodeficiency virus (HIV) and influenza A and B. The advantage of these tests is their ease of use and rapid results, usually in less than 30 minutes. However, the HIV rapid test relies on the detection of antibodies generated by the patient against HIV, which can take several months to reach a detectable level. In line with this, these tests are only qualitative in nature and have been shown to have a high occurrence of false positive and false negative results.
Therefore, there is a need for sensitive, quantitative detection of viral particles in a rapid point-of-care protocol amenable to self-administration with small sample volumes.